Until now, there has been no direct evidence on the mechanisms by which a cell can generate membrane. This project, while set out to unravel the mechanism of secretion, has uncovered one of the most fundamental processes in biology -- membrane biogenesis and membrane fusion. It not only offers a new perspective on how a cell can rapidly assemble new membrane from stored membrane precursor elements, it also resolves many unanswered questions concerning exocytosis. This project therefore, spans two very fundamental areas: membrane, without it cellular life would be impossible; and exocytosis, the mechanism by which cells secrete, communicate, and regulate. The significance of this project to biomedical research and the mission of the Institute is self-evident. This project has now been terminated at NIH and is being conducted at AFRRI. We have established our hypothesis of de novo membrane generation on much firmer ground by accomplishing the following: (1) We have demonstrated by serial sections and by the principle of metachromasy that the extensive expansion of secretory perigranular membrane can occur prior to its fusion with the plasma membrane. This set the stage for the requirement of the occurrence of a rapid new membrane assembly and membrane insertion into the perigranular membrane as part of the mechanism for granule exocytosis. (2) We have succeeded in isolating quiescent secretory granules from rat mast cells and have determined their phospholipid contents. The results indicate that the granule contains enough phospholipid alone to quadruple the surface area of the perigranular membrane. (3) We have found evidence that new membrane assembly also occurs in A23187 induced mast cell granule exocytosis. (4) Preliminary experiments on antigen challenge of IgE-presensitized mast cells also show the occurrence of de novo membrane generation, suggesting that this rapid process of membrane biogenesis is an integral part of the mechanism of exocytosis.